A database of circadian and diel rhythmic gene expression in the yellow fever mosquito Aedes aegypti

Abstract

Background: The mosquito species Aedes aegypti is the primary vector of many arboviral diseases, including dengue and yellow fevers, that are responsible for a large worldwide health burden. The biological rhythms of mosquitoes regulate many of the physiological processes and behaviors that influence the transmission of these diseases. For insight into the molecular basis of biological rhythms, diel and circadian gene expression profiling has been carried out for many species. To bring these resources to Aedes aegypti researchers, we used microarray technology to carry out a genome wide assessment of gene expression during the 24 hour light/dark (LD) cycle and during constant darkness (DD). The purpose of this report is to describe the methods, the validation of the results, and the organization of this database resource.

Description: The Aedes aegypti Circadian Database is a publicly accessible database that can be searched via a text-based query to visualize 44 hour temporal expression patterns of a given gene in Ae. aegypti heads under diel (observed under a 12 hour/12 hour LD cycle) and circadian (observed under DD) conditions. Profiles of gene expression under these conditions were assayed by Nimblegen 12-plex microarrays and rhythmicity was objectively assessed by the JTK_CYCLE algorithm. The output of the search is a graphical representation of the expression data along with computed period length, the time-of-day of gene expression peaks, and statistical determination for rhythmicity.

Conclusion: Our results show that at least 7.9% of the gene set present in the Aedes aegypti head are rhythmic under LD conditions and 6.7% can be considered circadian, oscillating under constant dark conditions. We present these results in the Aedes aegypti Circadian Database through Bioclock, a public website hosted by the University of Notre Dame at http://www.nd.edu/~bioclock/. This website allows searchable browsing of this quantitative gene expression information. The visualization allows for gene-by-gene comparison of transcript expression under both diel and circadian conditions, and the results are presented graphically in a plot profile of gene expression. The Ae. aegypti Circadian Database provides a community resource for observing diel and circadian fluctuations in gene expression across the Ae. aegypti genome.

Figure 1

Venn diagram showing the number of rhythmic genes observed under LD and DD conditions. The Venn diagram depicts different classes of genes in the mosquito head, including rhythmic only under LD conditions, rhythmic only under DD conditions, rhythmic under both LD and DD conditions, and genes not scored as rhythmic.

Figure 2

Heatmap of genes found to be rhythmic under LD (left) and DD (right) conditions. Images show the hierarchical clustering of genes found to be rhythmic. The time points taken during the 44 hour experimental period are shown above the images, and horizontal black/white or black bars below the images indicate the day/night or constant dark conditions. An expression intensity key is displayed in lower right. Yellow represents higher expression, and blue represents lower expression, relative to the mean expression value for each gene.

Figure 3

Expression profiles of clock genes determined by qPCR verify microarray analysis. Individual profiles of microarray data demonstrate diel and circadian profiles of a set of clock genes (A). These profiles were then verified by qRT-PCR (B). Cycle gene (aeCYC, circadian protein clock/arnt/bmal/pas, AAEL002049) is the ortholog of An. gambiae cycle (CYC, AGAP012873) and Drosophila cycle (cyc); and cryptochrome 2 (aeCRY2, DNA photolyase, AAEL002602) is the ortholog of An. gambiae cryptochrome 2 (CRY2, AGAP004261). Values are normalized by median fold change across the time course. Description of genes and primers used for qPCR are in Table  1. Horizontal black/white or black bars represent the day/night or constant dark collection conditions.

Figure 4

Screenshot of the Aedes aegypti Circadian Database demonstrates the key features of the user interface. In this example, tools are being utilized to identify the period gene (AAEL008141). A. On click, the search function allows for querying of gene names, gene descriptions and assigned AAEL numbers. B. The “mouseover” of each individual data point provides specific values for: (1) Timepoint, time (in hours) of collection of sample after onset of experiment (at ZT or CT 0), (2) LD/DD, light/dark or constant dark conditions of the sample, (3) Expression Value, raw fluorescence intensity of the data point, (4) Probability (p) value, for the individual gene that determines likelihood of rhythmicity (p <0.05 assigned as rhythmic), (5) False discovery rate (FDR) q-value for each gene (6) Amplitude, a measure of the maximum extent of oscillation of each optimal cyclical pattern, (7) Peak, time of day a gene was determined to have its highest intensity based on the cosine wave fit, and (8) Period, the time interval between one event in the cycle (e.g. peak or trough) in expression and the next sequential event in expression. The JTK_CYCLE algorithm was used to calculate the probability, amplitude, peak and period values. C. Image, Data, Crosstab, or PDF files can be generated and downloaded from the database. To download the data and visualizations for a given profile, click the download button depicted as a right facing arrow in an open box. Immediately to the right of the download button is a refresh button, identifiable as the circular left-facing arrow, that resets the database to its default settings. D. Double clicking on the profile screen allows the user to zoom in on a profile for close examination of profiles. Clicking the home button returns the screen to the original image.